The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis

Abstract

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

Keywords: HMGB1; caspase-11; endotoxemia; inflammasome; pyroptosis; sepsis.

Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.

Copyright © 2018 Elsevier Inc. All rights reserved.

Figures

Figure 1.. HMGB1 Enables Extracellular LPS to Activate Caspase-11 through Physical Binding to LPS

(A) Mice received i.p. biotin-labeled LPS (5 mg/kg) or normal saline (NS). Two hours later, peritoneal lavage fluid was incubated with streptavidin-coated beads. Pull down of HMGB1 was revealed by immunoblots. (B) Immunoblots for caspase-11, IL-1α, caspase-1, IL-1β, and β-actin in the supernatants (SN) or cell lysates (cell) of mouse peritoneal macrophagesstimulated with the indicated LPS-interacting proteins (LIPs, 1 μg/mL) plus LPS (1 μg/mL) for 16 hr. (C) LDH assay and ELISA for TNF in the supernatants of mouse peritoneal macrophages stimulated with LPS alone (1 μg/mL) or LPS (1 μg/mL)+HMGB1 (400 ng/mL) for 16 hr. (D) Immunoblots for caspase-11, IL-1α, caspase-1, IL-1β, and β-actin in the supernatants (SN) or cell lysates (Cell) of WT or Casp11−/− peritoneal macrophages stimulated with LPS alone (1 μg/mL) or LPS (1 μg/mL)+HMGB1 (400 ng/mL) for 16 hr. (E) ELISA for total IL-1α and LDH assay in the supernatants of WT or Casp11−/−peritoneal macrophages stimulated with LPS alone (1 μg/mL) or LPS (1 μg/mL)+HMGB1 of indicated concentration for 16 hr. (F) Immunoblot to detect caspase-11, IL-1α, caspase-1, IL-1β, HMGB1, and β-actin in supernatants (SN) or cell lysates (Cell) of mouse peritoneal macrophages of indicated genotypes upon exposure to the necrotic Hmgb1−/− or Hmgb1+/+ MEFs (106 cells/mL) in the presence or the absence of LPS (1 μg/mL) for 16 hr (the ratio of necrotic cells to macrophages is 1:1). (G) Immunoblot to detect caspase-4 (Casp4), IL-1α, caspase-1, IL-1β, and β-actin in supernatants (SN) or cell lysates (Cell) of human monocytic THP-1 cells transfected with scrambled siRNA or CASP4-specific siRNA upon HMGB1 (400 ng/mL) and LPS (1 μg/mL) stimulation for 16 hr. (H) LDH assay in the supernatants of human monocytic THP-1 cells transfected with scrambled siRNA or CASP4-specific siRNA upon HMGB1 (400 ng/mL) and LPS (1 μg/mL) stimulation for 16 hr. (I) LDH assay for WT or Casp11−/− mouse lung endothelial cells stimulated with LPS (1 μg/mL) alone or HMGB1 (400 ng/mL) alone or LPS (1 μg/mL)+HMGB1 (400 ng/mL) for 16 hr. (J) The LPS-binding capacity of HMGB1 incubated with different concentrations of LPS-RS. Plates coated with recombinant HMGB1 (16 μg/mL) were incubated with biotin-labeled LPS (0.2 μg/mL) with indicated concentration of LPS-RS. Binding between plate-coated HMGB1 and biotin-labeled LPS was measured by using streptavidin-HRP. The percentage of binding competition by LPS-RS was determined. (K) Mouse peritoneal macrophages were stimulated with HMGB1 (400 ng/mL) and LPS (1 μg/mL) in the presence of indicated doses of LPS-RS for 16 hr. Caspase-11, IL-1α, caspase-1, IL-1β, and β-actin were detected by immunoblot. (L) LDH assay for cytotoxicity and ELISA for IL-1α in the supernatants of WT mouse peritoneal macrophages stimulated with LPS (1 μg/mL)+HMGB1 (400 ng/mL) in the presence of different concentrations of HPep1 for 16 hr. Graphs show the mean ± SD of technical replicates and are representative of at least three independent experiments. See also Figures S1 and S2

Figure 2.. HMGB1 Delivers Extracellular LPS into the Cytosol through RAGE-Mediated Internalization

(A) Immunoblot for Na+-K+ ATPase, Rab7, Lamp1, and β-actin (left) and LPS activity assay (right) in the cytosolic and residual fraction (including cytoplasmicmembranes, endosomes, lysosomes, nuclei, etc.) from LPS (L, 1 μg/mL)- or LPS (1 μg/mL)+HMGB1 (400 ng/mL) (LH)-stimulated mouse peritoneal macrophages. (B) The physical interaction between caspase-11 and LPS were visualized as the red spots by PLA in mouse peritoneal macrophages primed with 100 ng/mL LPS for 4 hr and then stimulated with LPS alone (L, 5 μg/mL), HMGB1 alone (10 μg/mL) (H), or LPS (5 μg/mL)+HMGB1 (10 μg/mL) (LH) for 2 hr. Scale bar: 10 μm. (C and D) LPS activity assay in the cytosolic fraction (C) or ELISA for total IL-1αand IL-1β in cell culture medium (D) of LPS (L, 1 μg/mL) or LPS (1 μg/mL)+HMGB1 (400 ng/mL) (LH)-stimulated mouse peritoneal macrophages either placed at 4 or 37 degrees for 2 hr or pretreated with 20 μM dynasore (LHD) for 0.5 hr and washed away before LH treatment. (E and F) LPS activity assay on the cytosolic fraction (E) or ELISA for IL-1α or LDH assay in cell culture medium (F) of LPS+HMGB1-stimulated peritoneal macrophages from WT or Ager−/− mice. (G–I) LPS activity assay on the cytosolic fraction (G and H) or LDH assay in cell culture medium (I) of WT, Ager−/−, or Casp11−/− mouse lung endothelial cellspretreated with 20 μM dynasore for 0.5 hr stimulated with LPS or HMGB1 with or without pre-incubation of 20 μM dynasore for 0.5 hr. Graphs show the mean ± SD of technical replicates and are representative of at least three independent experiments. See also Figure S3.

Figure 3.. HMGB1 Destabilizes Lysosomal Membranes Leading to LPS Release into the Cytosol

(A) Confocal microscopy of mouse peritoneal macrophages incubated with fluorescent dextran (red) or DQ ovalbumin (red) alone or together with HMGB1 (5 μg/mL) for 4 hr then fixed and stained with DAPI (blue). Scale bar: 10 μm. (B) Confocal microscopy of WT or Ager−/− mouse peritoneal macrophages incubated with fluorescent dextran (red) alone or together with HMGB1 (5 μg/mL) or LBP (5 μg/mL) for 4 hr then fixed and stained with DAPI (blue). Scale bar: 10 μm. (C) Immunoblot for Cathepsin D, Na+-K+ ATPase, Rab7, Lamp1, and β-actin in the cytosolic fraction from vehicle-treated (C) or HMGB1 (H, 5 μg/mL)- or LBP (5 μg/mL)-stimulated WT or Ager−/− mouse peritoneal macrophages. (D) Flow cytometry of WT or Ager−/− mouse peritoneal macrophages stained with acridine orange and then treated with HMGB1 (5 μg/mL) or LBP (5 μg/mL) for 6 hr. (E) Dynamic imaging of HMGB1 protein labeled with Alexa Fluor 488 (1 μg/mL) on living cell membranes. Scale bar: 2 μm. (F) Whole-cell patch-clamp recording of HMGB1-induced inward current across the cytoplasmic membrane in proximity to the patch-clamp of HEK293 cells at neutral (pH = 7.4) or acidic (pH = 5.0) conditions. (G) Fluorescent calcein dye was encapsulated into the liposomes, which were incubated with HMGB1, bovine serum albumin (BSA), or TAT at the indicated concentration for indicated time at neutral (pH = 7.4) or acidic pH (pH = 5.0). Liposome leakage was monitored by measuring calcein fluorescence intensity. Triton X-100 treatment was used to achieve 100% liposome leakage. Graphs show the mean ± SD of technical replicates and are representative of three independent experiments. See also Figure S3.

Figure 4.. Hepatocyte-Released HMGB1 Is Critical for Caspase-11-Dependent Pyroptosis and Lethality in Endotoxemia

(A–E) Plasma HMGB1, IL-1α, and LTB4 concentrations from mice of indicated genotypes injected with low dose of LPS (400 μg/kg) for 6 hr and then challenged with high dose of LPS (10 mg/kg) for 4 hr. (F and G) Kaplan-Meier survival plots for mice with indicated genotypes injected with low dose of LPS (400 μg/kg) for 6 hr and then challenged with high dose of LPS (10 mg/kg) with or without SC-560 (5 mg/kg). (H) Plasma HMGB1 concentrations from mice of indicated genotypes injected with poly(I:C) (10 mg/kg) for 6 hr. (I–K) Plasma IL-1α or LTB4 concentrations from mice of indicated genotypes injected with poly(I:C) (10 mg/kg) for 6 hr and then challenged with LPS (10 mg/kg) for 4 hr. (L) Kaplan-Meier survival plots for mice with indicated genotypes injected with poly(I:C) (10 mg/kg) for 6 hr and then challenged with LPS (10 mg/kg). Circles represent individual mice.∗p < 0.05;∗∗p < 0.01;∗∗∗p < 0.001; NS: not significant (Student’s t test and log-rank test for survival). See also Figure S4.

Figure 5.. Neutralizing Extracellular HMGB1 or Deletion of Ager Prevents Pyroptosis and Lethality in Endotoxemia

(A) Plasma IL-1α concentrations from WT mice injected with low-dose LPS (400 μg/kg) for 6 hr and then subjected to administration of high-dose LPS (10 mg/kg) with monoclonal HMGB1 neutralizing antibody (2G7, 150 μg per mouse) or isotype control IgG (15 μg per mouse) for 4 hr. (B and C) Kaplan-Meier survival plots for WT mice injected with low-dose LPS (400 μg/kg) (B) or poly(I:C) (10 mg/kg) (C) for 6 hr and then subjected to administration of a high-dose LPS (10 mg/kg) with monoclonal HMGB1 neutralizing antibodies (2G7, 150 μg per mouse) or isotype control IgG (150 μg per mouse). (D and E) Plasma IL-1α or LTB4 concentrations from WT or Ager−/− mice injected with low-dose LPS (400 μg/kg) for 6 hr and then challenged with high-dose LPS (10 mg/kg) for 4 hr. (F) Kaplan-Meier survival plots for WT or Ager−/− mice injected with a low dose of LPS (400 μg/kg) for 6 hr and then challenged with a high dose of LPS (10 mg/kg). (G and H) Plasma IL-1α or LTB4 concentrations from WT or Ager−/− mice injected with poly(I:C) (10 mg/kg) for 6 hr and then challenged with LPS (10 mg/kg) for 4 hr. (I) Kaplan-Meier survival plots for WT or Ager−/− mice injected with poly(I:C) (10 mg/kg) for 6 hr and then challenged with LPS (10 mg/kg). Circles represent individual mice.∗p < 0.05;∗∗p < 0.01;∗∗∗p < 0.001; NS: not significant (Student’s t test and log-rank test for survival). See also Figure S4.

Figure 6.. Caspase-11, Hepatocyte-Released HMGB1, and RAGE Are Critical for Pyroptosis and Lethality in Bacterial Sepsis

(A–F) Plasma HMGB1, IL-1α, IL-1β, and LTB4 concentrations from mice of indicated genotypes were subjected to either cecum ligation and puncture (CLP) or sham operation. (G) Cell death in the spleens of indicated transgenic mouse strains was assessed by TUNEL staining. Shown in the right panel is the percentage of TMR-positive cells. Scale bar: 20 μm. (H–J) Kaplan Meier survival curves for the indicated transgenic mouse strains subjected to CLP. Circles represent individual mice.*p

Figure 7.. Hepatocytes Release HMGB1 in Response to LPS or Poly(I:C) by Distinct Mechanisms

(A) HMGB1 release from isolated primary WT or Tlr4−/− hepatocytes stimulated by LPS (0.1 μg/mL) for 16 hr. (B) Primary hepatocytes isolated from mice of indicated genotypes co-cultured with WT mouse peritoneal macrophages are shown in the left panel. IL-1α released from macrophages after stimulation of LPS (0.1 μg/mL) is shown in the right panel. (C and D) Plasma HMGB1 and IL-1α concentrations from mice of indicated genotypes injected with a low-dose LPS (400 μg/kg) for 6 hr and then challenged with high-dose LPS (10 mg/kg) for 4 hr. (E) Kaplan-Meier survival plots for mice with indicated genotypes injected with low-dose LPS (400 μg/kg) for 6 hr and then challenged with high-dose LPS (10 mg/kg). (F) LPS activity assay of the cytosolic fraction from LPS (0.1 μg/mL)-stimulated primary hepatocytes from indicated transgenic mouse strains. (G) Primary hepatocytes isolated from mice of indicated genotypes were stimulated by LPS alone (100 ng/mL) or LPS+poly(I:C) (PIC) (10 μg/mL) for indicated time. Then the expression of Caspase-11 and GAPDH were determined by immunoblot. (H) HMGB1 release from isolated primary WT or Casp11−/− hepatocytes stimulated by LPS (0.1 μg/mL) for 16 hr. (I–K) Plasma HMGB1 or IL-1α concentrations from mice of indicated genotypes injected with low-dose LPS (400 μg/kg) for 6 hr and then challenged with LPS (10 mg/kg) for 4 hr. (L) Kaplan-Meier survival plots for mice with indicated genotypes injected with low-dose LPS (400 μg/kg) for 6 hr and then challenged with LPS (10 mg/kg). (M) HMGB1 release from isolated WT, Tlr4−/−, Casp11−/−, Tlr4−/−Casp11−/−, Gsdmd−/−, or Ifnar1−/− hepatocytes stimulated by poly(I:C) (10 μg/mL) for 24 hr. (N) Plasma HMGB1 concentrations for Tlr4−/− mice injected with poly(I:C) (10 mg/kg) for 6 hr. (O) Kaplan-Meier survival plots for Tlr4−/− mice injected with poly(I:C) (10 mg/kg) for 6 hr and then challenged with LPS (10 mg/kg) with monoclonal HMGB1 neutralizing antibodies (2G7, 150 μg per mouse) or isotype control IgG (150 μg per mouse). *p